Milk products such as e.g. fermented milk products (e.g. yogurt) are well known in the art.
As known in the art—properties such as viscosity and gel firmness are important properties for relevant milk products.
Generally speaking—if one can make more viscosity and/or gel firmness, using the same milk composition, one can make the same viscosity/gel firmness using less milk (or other) solids, thus saving on raw material (or obtaining a higher yield).
For low fat fermented milk products such as e.g. low fat yogurt it may be difficult to obtain an optimal preferred texture of the products—more precisely the problem may be to e.g. get a sufficient high texture viscosity in such low fat products.
Addition of protein, typically skim milk powder or whey based proteins, may be seen as a standard procedure to improve the texture in low fat yogurts. However, such a solution may be costly and does not fully comply with the concept of a low fat/low calories product since the added protein also contributes to the total energy content.
In relation to improvement of the texture—US2005/0095316A1 paragraph [0005] describes adding so-called texturing agents (thickeners, gelling agents) such as starch, pectin or gelatin. However, as known—to e.g. have extra pectin or gelatin in e.g. a yogurt product may not be preferred.
Within the last years so-called texturizing cultures, based on extracellular polysaccharides (EPS) production, have markedly improved the viscosity of such low fat milk products—for instance WO2007/095958A1 (Chr. Hansen A/S) describes that strains of Streptococcus thermophilus synthesize EPS that may give a desirable “ropy” or viscous texture to fermented milk products.
Accordingly, one may say that for e.g. low fat yogurt is the texture viscosity problem today as such pretty well solved by use of e.g. these EPC producing cultures.
However, as described in the article of A. N. Hassan et al (J. Dairy Sci: 86:1632-1638; 2003) the so-called gel firmness may decrease significantly when using such EPS producing cultures as compared to non EPS producing cultures.
Accordingly—one may say that the situation today is that for e.g. low fat yogurt products have the earlier viscosity problem essentially been solved by the use of the EPS producing cultures—however, the use of the EPS cultures may have “created” a new problem in relation to the decreased gel firmness.
In the article of A. N. Hassan et al is in the abstract referred to lower viscoelastic moduli—as known to the skilled person this viscoelastic moduli feature relates to gel firmness, in the sense that if the product has lower viscoelastic moduli it will have lower gel firmness.
In FIG. 1 of the article is shown that that shear stress of the EPS yogurts was much higher, which to a person skilled in the art directly translates to a higher viscosity.
As discussed above—for milk products in general such as e.g. fermented milk products (e.g. yogurt)—the so-called gel firmness is a very relevant property.
For instance, low gel firmness may give an undesirable mouth feel to the milk product.
Further, if e.g. a yogurt has a low gel firmness is will appear thin and flow too readily—e.g. on the spoon or e.g. in a bowl.
Further, if e.g. a yogurt has a low gel firmness it may be prone to syneresis (see below) due to whey separation.
As is well known in the art, milk clotting enzymes, such as the protease chymosin (alternatively named rennin) is used to make cheese, where chymosin causes coagulation and curd formation.
As is well known cheese making comprises three steps, or stages, that all come about as the result of addition of a milk clotting enzyme: 1) the formation of a clot (or soft gel), the solidification, or firming of this clot, and the eventual expulsion of whey, the latter process also called syneresis.
As evident—when making e.g. a yogurt one is generally not interested in getting this syneresis effect—i.e. the separation of the milk into solid curds and liquid whey.
Accordingly, use of a milk clotting enzyme like chymosin is generally not preferred when one wants to make e.g. a yogurt.
US2005/0095317A1 (Danone) describes the use of a protease with kappa-caseinolytic activity in the production fermented milk products such as yogurt.
The protease may e.g. be chymosin—see e.g. [0028] of the US patent application.
The proteases (e.g. chymosin) hydrolyse casein in the milk—accordingly one should prima facie have believed that the proteases (e.g. chymosin) could have given rise to a for yogurt production unwanted syneresis effect.
However, section [0008] explains that surprisingly and unexpectedly it was shown that use of proteases such as e.g. chymosin “improve the texture, and in particular to increase the viscosity of yogurts and fermented milks, without as a result inducing syneresis which would be unacceptable for such fermented dairy products”.
US2005/0095316A1 (Danone) essentially relates to the same technical teaching as in US2005/0095317A1 (Danone) discussed above—however, in this US application the relevant proteases are defined as bacterial proteases (chymosin is from cows—i.e. it is not a bacterial protease).
U.S. Pat. No. 7,560,127B2 (DSM) describes use of special deglycosylation enzymes in cheese production. The deglycosylation enzymes are defined as enzymes that can deglycosylate the kappa casein (κ-casein) present in the milk. As discussed below—casein is a protein with so-called O-linked glycosylation. Accordingly, the deglycosylation enzymes mentioned in this US patent are enzymes that can deglycosylate O-linked glycosylated proteins such as kappa-casein (kappa-casein is a so-called O-linked glycoprotein).
The US patent reads on column 1, lines 51-58:
“It was surprisingly found that a deglycosylation of κ-casein will lead to clotting as it are the sugars associated with κ-casein that carry the negative charge which stabilize the casein micelles. Clotting of the milk in this way results in a process in which a larger part of the κ-casein is retained in the cheese and a higher yield can be obtained than using proteolytic activity of chymosin.”
In short, one may say that this U.S. Pat. No. 7,560,127B2 patent essentially describes that one may get the, for cheese production, required clotting by using the mentioned O-linked related deglycosylation enzymes instead of chymosin. Since this U.S. Pat. No. 7,560,127B2 patent relates to production of cheese and in the claim 1 it is said that one may get the cheese without using a protease (such as chymosin) the skilled person will implicitly understand that the use of the O-linked related deglycosylation enzymes must lead to a substantial amount of syneresis.
It is here relevant to note that U.S. Pat. No. 7,560,127B2 relates to production of cheese, where a substantial amount of syneresis is required. Consequently, a skilled person would not expect it to apply to fermented milk product like yogurt, where syneresis is generally highly undesirable.
Further, U.S. Pat. No. 7,560,127B2 does not explicitly say anything about the herein relevant gel firmness property—i.e. one may say it only explicitly relates to the clotting/syneresis effect attributed to use of the described O-linked deglycosylation enzymes instead of chymosin.
The article of E. Cases et al (Journal of Food Science; Vol. 68, Nr. 8, 2003, Pages 2406-2410) describes deglycosylation of chemically acidified milk with an O-linked deglycosylation enzyme (neuraminidase; EC 3.2.1.18).
The milk is chemically acidified with the chemical GDL (see p. 2407, section “Acid milk coagulation”)—accordingly, there is in the E. Cases et al article not described a “fermented milk” product inoculated with relevant microorganisms (e.g. a yogurt).
The article of E. Cases et al describes that the use of the O-linked deglycosylation enzyme neuraminidase (EC 3.2.1.18) gave higher final gel firmness to the enzymatically treated and chemically acidified milk.
It is here relevant to note that the article of E. Cases et al does not say anything of herein relevance with respect to the possible syneresis effect of using the O-linked deglycosylation enzyme neuraminidase (EC 3.2.1.18).
In the E. Cases et al article is not provided relevant evidence for the purity of the used neuraminidase (EC 3.2.1.18) O-linked deglycosylation enzyme preparation.
In view of this—it is submitted, that the enzyme preparation used may have comprised some relevant protease enzyme activity and that this protease enzyme activity could have been responsible for the described gel firmness effect—as discussed above, US2005/0095317A1 (Danone) describes that the use of a protease may improve the gel firmness.
The relevance of this suspicion is further underlined by that fact that Clostridium perfringens, the microorganism that the neuraminidase in the E. Cases et al. article was derived from (see Materials and Method section) is known to contain more than 140 proteolytic enzymes—see e.g. the authoritative Merops peptidase database (http://merops.sanger.ac.uk/cgi-bin/speccards?sp=sp000283;type=peptidase).
With no information on purity of the enzyme preparation provided, it thus seems very plausible that it was not purified to the exclusion of all relevant proteolytic enzymes that could in themselves have had the effect on gel firmness described in the E. Cases et al article.
As known in the art—the O-linked deglycosylation enzyme neuraminidase (EC 3.2.1.18) may also have some N-linked glycosidase activity. However, the E. Cases et al article discussed above only refers to the O-linked glycosidase activity of neuraminidase (EC 3.2.1.18) when herein relevant enzymatic activities of neuraminidase is discussed in he E. Cases et al article.
EP1489135A1 may be seen as relating to use of deglycosylated oleuropein (obtained from Olive leaf extracts) for increasing gel firmness of acidified whey milk and yogurt. Beta-glycosidase (beta-1,6-glucosidase) and lactase are in this document “simply” used to deglycosylate the oleuropein—i.e. the enzymes are here not the active component for increasing the gel firmness in the milk/yogurt. Oleuropein is not a protein/peptide with enzymatic activity. Beta-glycosidase is neither an N- nor O-linked glycosidase. The method of the present invention does preferably not comprise addition of an activated olive leaf extract (or other extracts) as disclosed in EP1489135A1 to an animal milk substrate.